Apr 14, 2020

Public workspaceMouse Pancreatic Islet Isolation

DOI
dx.doi.org/10.17504/protocols.io.sqaedse
  • 1University of Alberta
  • CIRTNR2FIC
Aliya F Spigelman
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DOI: dx.doi.org/10.17504/protocols.io.sqaedse
External link: http://www.bcell.org
Protocol Citation: Nancy Smith, Aliya F Spigelman, Haopeng Lin, Patrick E Macdonald 2020. Mouse Pancreatic Islet Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.sqaedse
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 17, 2018
Last Modified: April 14, 2020
Protocol Integer ID: 14818
Keywords: islets, isolation, pancreas, mouse, perfusion, purification, collagenase
Abstract
This protocol details islet isolation from mouse pancreas. The protocol is divided into 3 main parts; in situ perfusion of pancreas with collagenase, pancreas digestion, and islet purification. A link is provided for the purification using histopque gradient.
Guidelines
Digestion time using collagenase from Clostridium histolyticum type V is lot specific. Lot #010M8620 has a 7 minutes digesting time. New lots require testing and digestion time must be adjusted for the new lot's activiity.
Materials
MATERIALS
ReagentFBS (Canadian Origin) Gibco - Thermo Fisher ScientificCatalog #12483-020
ReagentSodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S5761
Reagent5cc syringesBecton Dickinson (BD)Catalog #302832
ReagentExtra fine Iris scissorsFine Science ToolsCatalog #14084-08
ReagentAdson serrated forceps Fine Science ToolsCatalog #11006-12
ReagentAdson 1x2 teeth forcepsFine Science ToolsCatalog #10027-12
ReagentCurved ForcepsFine Science ToolsCatalog #11001-12
ReagentHalsted-Mosquito hemostatsFine Science ToolsCatalog #13009-12
ReagentUltra Fine point forcepsFine Science ToolsCatalog #11370-40
ReagentGibco Penicillin-Streptomycin (10000 U/ml)Fisher ScientificCatalog #15-140-122
Reagent30G needlesBecton Dickinson (BD)Catalog #B305106
ReagentGibco RPMI 1640Thermo Fisher ScientificCatalog #11875
ReagentHanks Balanced Salts (HBSS)Merck MilliporeSigma (Sigma-Aldrich)Catalog #H6136
ReagentCollagenase from Clostridium histolytic type V lot #010M8620Merck MilliporeSigma (Sigma-Aldrich)Catalog #C9263
Before start
Hanks’ Balanced Salts (HBSS) and Mouse Islet Culture Media should be prepared in advance.
Solution Prep- Hanks’ Balanced Salts (HBSS) - Sigma H6136
Solution Prep- Hanks’ Balanced Salts (HBSS) - Sigma H6136
Measure out 900ml of room temperature H2O.

While gently stirring the water, add the powdered medium. Stir until dissolved. DO NOT HEAT.
Rinse original vial with water to remove traces and add to above.
Add 0.35g sodium bicarbonate and stir until dissolved.
Amount0.35 g Sodium Bicarbonate
Adjust pH to 7.4
Bring solution to 1L.
Store at 4°C
Temperature4 °C
Soultion Prep - Mouse Islet Culture Media
Soultion Prep - Mouse Islet Culture Media
Add FBS and Pen/Strep to bottle of RPMI

500ml RPMI 1640 (11.1mM glucose)Gibco 11875-119
50ml FBS Canadian Origin Gibco 12483-020
5ml Pen/strep (10000 Unit/ml/10000 ug/ml)Gibco 15140-122

Store at 4°C
Temperature4 °C

Solution Prep- Collagenase type V – Sigma C9263 lot 010M8620:
Solution Prep- Collagenase type V – Sigma C9263 lot 010M8620:
Dissolve 1mg/ml Collagenase in HBSS from above.
  • Approximately 5ml per mouse for injection and shaking
  • Make fresh before isolation, and keep on ice (use within the hour)
Pancreas Perfusion
Pancreas Perfusion
Euthanize mouse according to your institute's research ethics protocols. (We use a CO2 chamber)
Make a midline incision from the lower abdomen to the sternum.
Common bile duct is tied or clamped where it meets the intestine.
Collagenase is injected in situ via the common bile duct using a size 30G needle until the pancreas is inflated (typically 1-2 mL). The head and the tail of the pancreas should be inflated to maximize the number of islets isolated.
The pancreas is removed and placed in tube (15mL or 50mL depends on preference)  with remaining collagenase (1-2mL) and kept on ice until the next step.
Digestion
Digestion

After isolation, allow digestion to proceed for 7 minutes (lot specific -Sigma C9263 lot 010M8620 ) in shaking water bath at 37°C.
Duration00:07:00
Temperature37 °C

Shake digested material vigorously.
Add Mouse Islet Media to stop digestion. Tubes remain on ice until ready for purification.


Purification
Purification
Allow islets to settle (2-3 minutes) and remove excess fat. Proceed to the histopaque gradient protocol to purify the islets. If hand picking is preferred, go to the next step.
https://www.protocols.io/view/purification-of-mouse-pancreatic-islets-using-hist-u7ueznw
Pour islets into a non tissue culture dish and pick islets into a 35mm non tissue culture dish.
Once islets picked cleanly, culture using Mouse Islet Media in 37°C, 5% CO2 until ready for use in experiment.